Updated project metadata. Cytomegalovirus (CMV) reactivation of latent infection following immune dysregulation remains a serious risk for patients, often causing significant morbidity and mortality. Herein, we demonstrate the CMV-encoded GPCR, US28, in coordination with cellular Ephrin receptor A2 (EphA2), attenuates MAPK signaling, thereby limiting viral replication in latently-infected primary monocytes. To define the underlying mechanism by which US28 modulates MAPK signaling during latency, we used two unbiased approaches to: 1) profile kinome changes in response to US28 expression, and 2) define US28 interacting partners that could influence its function(s). First, we evaluated changes in the cellular kinome in response to US28 expression in THP-1 monocytic cells compared to control cells using multiplexed kinase inhibitor beads coupled with mass spectrometry (MIB-MS) to quantitatively measure kinase expression and abundance. Next, we leveraged proximity labeling technology, whereby we inserted the biotin ligase gene, birA, conjugated to a C-terminal hemagglutinin (HA) epitope tag in-frame with the US28 ORF to generate TB40/E-mCherry-US28-bioID-HA. We then infected fibroblasts with US28-bioID-HA, added biotin 18 h prior to harvesting the cells at 48 h post-infection (hpi), and analyzed the biotin-conjugated proteins following streptavidin capture by affinity purification-mass spectrometry (AP-MS).